Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Detection and determination of nitrate and nitrite: a review.

Summary
Results from the pioneering research on the interactions between pH and denitrification in soil from the 1950s to the present are reviewed, the changing perceptions of this complex relationship are discussed, and the current status of the subject is assessed. Facets of this relationship that are analysed in detail include the direct or indirect influence of pH on overall denitrification rates in soils, changes in the composition of gaseous products that depend on pH, methods for measuring the process, the concept of an optimum pH for denitrification, and the adaptation of microbial denitrifying communities to acidic environments. The main conclusions to be drawn are as follows. Total gaseous emissions to the atmosphere (N2O, NO and N2) have repeatedly been shown to be less in acidic than in neutral or slightly alkaline soils. This may be attributable to smaller amounts of organic carbon and mineral nitrogen available to the denitrifying population under acid conditions rather than a direct effect of low pH on denitrification enzymes. Numerous laboratory and field studies have demonstrated that the ratio N2O:N2 is increased when the pH of soils is reduced. The relation between soil pH and potential denitrification as determined by various incubation methods remains unclear, results being influenced both by original conditions in soil samples and by unknown changes during incubation. The concept of an optimum pH for denitrification has been frequently proposed, but such a term has little or no meaning without reference to specific attributes of the process.

The influence of soil pH on denitrification: progress towards the understanding of this interaction over the last 50 years

Denitrification in recirculating systems: Theory and applications

Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N2O and N2 from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with 15N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N2O, and finally N2O was converted to N2. At pH 8.5, nitrate was converted directly to N2, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

Recording the amount of titrant required to maintain constant pH in a bioreactor where cell metabolism causes acidity changes allows on-line determinations of growth kinetics in computer-controlled batch cultures. A system for making such measurements is described and its performance is investigated. Transient bicarbonate accumulation occurs if the culture produces CO2 at high pH values and low gas transfer rates. We have developed a mathematical model for the titrant requirement as a function of the cell growth rate, the gas transfer properties of the bioreactor and the culture pH. According to this model, bicarbonate accumulation affects the stoichiometry between titrant and biomass but does not prevent determination of growth rate constants. These predictions are confirmed using model experiments and measurements during batch growth of microbial cultures.

On-line growth measurements in bioreactors by titrating metabolic proton exchange

The effects of oxygen on the use of nitrate as an electron acceptor by the denitrifying bacterium Thiosphaera pantotropha were investigated during growth on acetate. In batch cultures under aerobic conditions nitrate was not utilised and the growth rate constant was 0.55 h−1. The corresponding value for growth on nitrate under anoxic conditions was 0.37 h−1. In acetate-limited continuous cultures with feedback control of the dissolved oxygen concentration, nitrate utilisation was totally inhibited by the lowest concentration of oxygen tested (22 μM). Carbon conversion efficiencies with acetate increased from 0.28 with nitrate to 0.44 with oxygen. The rates of nitrification calculated from nitrogen balance studies were not greater than 1.5% of the rate of anoxic denitrification.

Jens Kirk Thomsen

Papers

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

On-line growth measurements in bioreactors by titrating metabolic proton exchange

Interactions between respiration and denitrification during growth of Thiosphaera pantotropha in continuous culture

Continuous turbidometric measurements of microbial cell density in bioreactors using a light-emitting diode and a photodiode

Alkanesulphonates as eluents for the determination of nitrate and nitrite by ion chromatography with direct UV detection